Methods for site-directed mutagenesis

Review these traditional PCR-based methods for creating a specific mutation in a known sequence, in vitro. Then read our follow-up article, Site-directed mutagenesis—Improvements to established methods (see the "Additional reading" sidebar) which describes how you can generate the same types of mutations, more quickly and efficiently, using custom, synthetic dsDNA fragments.

Site-directed mutagenesis is an in vitro method for creating a specific mutation in a known sequence. While often performed using PCR-based methods, the availability of custom-designed, synthetic, double-stranded DNA (dsDNA) fragments can drastically reduce the time and steps required to obtain the same sequence changes.

In this article we describe several PCR-based methods for site-directed mutagenesis. Primers designed with mutations can introduce small sequence changes, and primer extension or inverse PCR can be used to achieve longer mutant regions. Using these site-directed mutagenesis techniques allows researchers to investigate the impact of sequence changes or screen a variety of mutants to determine the optimal sequence for addressing the question at hand. The IDT Mutagenesis Application Guide provides more details on these approaches.

Traditional PCR

When PCR is used for site-directed mutagenesis, the primers are designed to include the desired change, which could be base substitution, addition, or deletion (Figure 1). During PCR, the mutation is incorporated into the amplicon, replacing the original sequence.

Mutations introduced by PCR can only be incorporated into regions of sequence complementary to the primers and not regions between the primers [1].

Figure 1. Site-directed mutagenesis by traditional PCR. Primers incorporating the desired base changes are used in PCR. As the primers are extended, the mutation is created in the resulting amplicon.

Primer extension

Site-directed mutagenesis by primer extension involves incorporating mutagenic primers in independent, nested PCRs before combining them in the final product [2]. The reaction requires flanking primers (A and D) complementary to the ends of the target sequence, and two internal primers with complementary ends (B and C). These internal primers contain the desired mutation and will hybridize to the region to be altered. During the first round of PCR, the AB and CD fragments are created. These products are mixed for the second round of PCR using primers A and D. The complementary ends of the products hybridize in this second PCR to create the final product, AD, which contains the mutated internal sequence (Figure 2A). Longer insertions can be incorporated by using especially long primers, such as IDT Ultramer™ oligonucleotides.

To create a deletion, the internal primers, B and C, are positioned at either side of the region to be deleted to prevent it from being incorporated within fragments AB and CD from the first round of PCR. The complementary sequences at the ends of the these fragments, created by primers B and C, enable hybridization of AB to CD during the second round of PCR, and the final product with the desired deletion (AD) is created (Figure 2B).

Figure 2. Site-directed mutagenesis by primer extension. (A) Insertion: Primers B and C contain the complementary sequence that will be inserted (blue line). Two reactions are performed in the first round of PCR using primer pairs A/B (1) and C/D (2). The resulting amplicons are mixed with primer pair A/D for the second round of PCR. The complementary ends of the first round amplicons hybridize and the PCR creates the final product with the desired insertion. (B) Deletion: Primers B and C are located on either side of the sequence to be deleted, and contain sequence from both sides of the deletion (black or gray additions that match the black or gray original sequence). Two reactions are performed for the first round of PCR using primer pairs A/B and C/D. The amplicons are mixed with primer pair A/D for the second round of PCR. The overlapping regions of these amplicons hybridize and the PCR creates the final product with the desired deletion.

Inverse PCR

Inverse PCR enables amplification of a region of unknown sequence using primers oriented in the reverse direction [3]. An adaptation of this method can be used to introduce mutations in previously cloned sequences. Using primers incorporating the desired change, an entire circular plasmid is amplified to delete (Figure 3A), change (Figure 3B), or insert (Figure 3C) the desired sequence.

Figure 3. Site-directed mutagenesis by inverse PCR. The primers used are 5’-phosphorylated to allow ligation of the amplicon ends after PCR. A high fidelity DNA polymerase that creates blunt-ended products is used for the PCR to produce a linearized fragment with the desired mutation, which is then recircularized by intramolecular ligation. (A) Deletion: Primers that hybridize to regions on either side of the area to be deleted are used. (B) Substitution: One of the primers contains the desired mutation (blue bubble). (C) Insertion: The primers hybridize to regions on either side of the location of the desired insertion (black, dotted line). One primer contains the additional sequence that will be inserted (blue line).